Polymers such as polysaccharides, polyvinyl alcohol and Nylon are widely used as solid supports for immobilizing enzymes and for preparing biospecific affinity matrices. Several methods are known for coupling biologically active ligands to water insoluble carriers. These methods have been used for the covalent immobilization of biologically active materials such as enzymes, antibodies or antigens. Immobilized biologically active materials find use in many different fields of technology. One example thereof is in immunologic determination methods. Another important application is in affinity chromatography, wherein an organic ligand having biospecific affinity to some other organic substance has been bonded to a water insoluble polymeric carrier. Proteins have been bonded to water soluble polymers as well as water insoluble polymers for modifying the properties of the protein.
Prior to development of the compositions and methods disclosed and claimed in Ser. No. 07/243,352, the entire disclosure of which is hereby incorporated by reference, no method had been found for preparing an activated polymeric support capable of effectively coupling a ligand at an acidic pH, for example at a pH less than about 4. Since some ligands must be coupled in the acidic pH range, the need arises for an activating agent which can produce activated polymers effective in the acid range of pH. Pepsin, for example, must be coupled at a pH of below about 4 because it is denatured and loses its enzymatic activity at a higher pH.
Ser. No. 07/243,352 has as its principal object methods and compositions for use in providing stable and hydrolysis-resistant coupling products of polymeric carriers and organic ligands. A particular object of that application is to provide chromatographic matrices which can be used to bind various organic molecules, and in particular biologically active materials, both covalently and non-covalently.
In accordance with the disclosure of Ser. No. 07/243,352 these and other objects are achieved by forming an "activated" derivative of a polymeric carrier by reacting the polymer with a substituted 2-halopyridine (as therein defined) in the presence of an organic base and then reacting the substituted polymer with a ligand containing one or more nucleophilic groups (such as amino, hydroxyl or sulfhydryl group). The method according to the invention of Ser. No. 07/243,352 thus comprises an activation step, wherein a substituted 2-halopyridine is introduced into a polymeric carrier, and a coupling step, in which an organic ligand is bonded covalently to the polymeric carrier. The preparation of several products believed generally useful in non-covalently binding organic molecules, and in particular proteins (such as serum albumin and immunoglobulins), is disclosed therein.
The development of compositions useful in the non-covalent binding of organic molecules is of tremendous significance to the chemical and biological arts, particularly when such compositions exhibit any degree of binding selectivity. For example, in spite of the widespread development and use of ion-exchange resins and Protein A affinity gels for separation and purification of organic materials of biological interest (such as, in particular, polyclonal and monoclonal antibodies as used in diagnostics, purification and experimental therapeutics), there remain several disadvantages inherent in these methods. The ion-exchange chromatographic method, due to its lack of specificity, requires in general the use of several columns and gradient elution procedures involving specific detection methods to monitor the process. On the other hand, while Protein A affinity gels are somewhat more specific, they are significantly more expensive than ion-exchange chromatography. Moreover, Protein A gels are unable to bind significant amounts of IgG from rats, goats or chickens. There is also the possibility of immune reactions due to Protein A inadvertently leaked into recovered antibody preparations, which may limit the value of chromatographic methods using Protein A gels for purification of materials with which binding does occur. Finally, immobilized Protein A gels are susceptible to microbial and protease degradation, as well as to other protein denaturing agents.
Therefore, it would clearly be desirable to develop a chromatographic support with a synthetic ligand, preferably one of low molecular weight and low cost, which would be capable of selectively and efficiently binding molecules of biological interest, and in particular, of binding antibodies from all species.
The development of thiophilic gels by Porath et al. is an effort directed toward this end [see, e.g., Porath J. et al, "A New Kind of `Thiophilic` Electron-Donor-Acceptor Adsorbent," Makromol. Chem., Macromol. Symp. 17: 359-71 (1988) and references cited therein]. In thiophilic interaction chromatography, antibody is adsorbed on a thiophilic gel in the presence of high concentrations of neutral water-structure-forming salts. Desorption is achieved by elution with buffer not containing the salts. The partial structure of a thiophilic gel may be illustrated as ##STR1## wherein represents the polymer backbone.
It is an object of the present invention to provide additional synthetic ligands capable of selectively and efficiently binding materials of interest from a variety of different sources.
The present invention has as its principal object providing a stable and hydrolysis-resistant coupling product of a polymeric carrier and an organic ligand.
Another object of the present invention is to provide such a process which can be conducted under relatively mild conditions in order to avoid any detrimental effect upon reactants such as sensitive biological ligands.
Another object of the present invention is to provide a series of activating agents which can be reacted with polymeric carriers to produce an activated polymer capable of being coupled to a wide variety of organic ligands at a wide pH range.
Another object of this invention is to provide chromatographic matrices which can be used to bind various biologically active materials both covalently and non-covalently.
It is also a further object of the present invention to develop materials capable of selective binding of antibodies from all species, including those from rats, goats and chickens heretofore not amenable to treatment using conventional purification methods employing Protein A affinity ligands.
It is yet a further object of the present invention to provide synthetic affinity ligands capable of adsorbing organic molecules of interest in general, and peptides such as serum proteins in particular, without the use of high salt concentrations in the adsorption process.
It is another object of the present invention to provide materials and processes for isolation and purification of immunoglobulins of various classes and from a variety of species without the use of proteinaceous materials which themselves could lead to contamination of the resulting product.
These and other objects of the invention are achieved in accordance with one aspect of the present invention by forming a reactive derivative of a polymeric carrier by reacting the polymer with a substituted 2-halopyridine as hereinafter defined and then reacting the activated polymer with a ligand containing one or more nucleophilic groups such as amino, hydroxyl or sulfhydryl group to form a coupled product.
The method according to this aspect of the present invention thus comprises an activation step, wherein a substituted 2-halopyridine is introduced into a polymeric carrier and a coupling step in which an organic ligand is bonded covalently to the polymeric carrier. The method may also comprise the use of the coupled product as a chromatographic matrix capable of binding various biologically active materials covalently or non-covalently, as desired.
The present invention is directed to a novel class of products, one method for the preparation of which is disclosed in Ser. No. 07/243,352, and to the use thereof in chromatographic methods for binding various biologically active materials non-covalently. In particular, the present invention is directed in another particular aspect thereof to the surprising discovery that members of a particular class of novel compounds which may be defined as the reaction product of a polymeric gel with a pyridine base, such as 4-dimethylaminopyridine (DMAP), and a halogen-substituted pyridine, such as 3,5-dichloro-2,4,6-trifluoropyridine (DCTFP), which reaction product may in turn optionally be reacted with other specified low-molecular-weight compounds or with a source of hydroxide ions, are capable of selectively and efficiently binding proteins and other organic materials of interest non-covalently to a degree comparable or superior to the heretofore preferred natural affinity ligands, such as Protein A gels.
In particular, it has now been determined that in accordance with one embodiment of the present invention, subsequent treatment of the reaction product of a Sepharose gel, DMAP and DCTFP with ethylene glycol leads to the formation of a product (hereinafter, "O-gel") which may be employed to achieve purification and recovery of proteins such as serum albumin and immunoglobulins of various classes from crude sources, such as serum samples from various species.
One type of method for the preparation of several members of this novel class of compounds is described in several examples of Ser. No. 07/243,352. The present invention is directed to attempts at elucidation of the structure of these compounds and exploitation of their exceptional utility in separation and purification of organic molecules, and in particular in the recovery of specific proteins in relatively pure form even from crude sources, such as serum samples.